The goal of this project is to provide information about the poly- beta-hydroxybutyrate biosynthetic pathway in Alcaligenes eutrophus H16 by obtaining, and analyzing, the DNA sequence of the entire pathway. The pathway is contained on a 5.2 kb KpnI/Eco RI fragment and has been cloned into plasmid pUC18. The pathway will be subcloned into plasmids pSP72 and pSP73 and a series of nested-set fragments will be generated from these plasmids using exonuclease III. The series of nested sets will be sequenced utilizing the SP6 and T7 promoters at either end of the inserts as primers. Sequence data from these gels will be analyzed for putative promoter sites, open reading frames, and various other prokaryotic consensus sequences using IBI/Pustell software. At the same time, the deletion set will be assayed for the three enzymatic activities of the PHB-biosynthetic pathway and this information will be used to locate the specific enzyme coding regions. Once the putative promoter site is located, another series of deletions will be generated that remove varying amounts of DNA in the promoter region. These deletions will be utilized to identify regions of DNA that specify the post- exponential induction of the PHB biosynthetic pathway. This data, in conjunction with data from another project, will be used to formulate a model for the PHB pathway and it's mechanism of transcription.